Carcinoembryonic antigens

ABSTRACT

Antigens associated with carcinomas and adenocarcinomas as well as methods for isolating, identifying and detecting them are disclosed.

United States Patent Hansen Feb. 18, 1975 [5 CARCINOEMBRYONIC ANTIGENS3,697,638 10/1972 Hansen 424/1 [76] Inventor: Hans John Hansen, 200Hillside OTHER PUBLICATIONS Allendaie 07401 Thomson et al., Proc. Natl,Acad. Sci. US. 64, [22] Filed: May 12, 1972 161-167, 1969. [21] AppL No;252,700 Kleist et al., Cancer Research, 29: 19614964, 1969.

Krupey et al., J. Exptl. Med., 128: 387-398, 1968. f Applicam Data Goldet al., J. Exptl. Med., 121-439-462, 1965 and [63] Continuatlon-ln-partof Ser. No. 133,404, Apr1l 12, 122: 467 481 1965 1971, Pat. No.3,697,638, which is a continuatiommpart of sen No 0,288, Jan 27,Freedman et 211., Can. Cancer Conf. 8: 407-424, l969 1971, abandoned,which is a continuation-in-part of Ser. No. 42,526, June 1, 1970,abandoned. Primary Examiner-l-1oward E. Schain Attorney, Agent, orFirmSamuel L. Welt; Jon S. [52] 11.8. Cl 260/112 R, 424/1, 424/12, S xGerald S. Rosen 424/85, 424/88, 23/230 B [51] Int. Cl. C07g 7/00, A6lk27/04 58 Field of Search 424/1, 12, 88; 260/112 R ABSTRACT Ant1gensassociated with carcinomas and adenocar- [5 References Ci d cinomas aswell as methods for isolating, identifying and detecting them arediSClOSGd.

3,663,684 5/1972 Freedman et a1 424/12 X 9 Claims, N0 Drawings 1CARCINOEMBRYONIC ANTIGENS RELATED APPLICATIONS BACKGROUND OF THEINVENTION The neoplastic process in human beings has been and still isthe subject of intensive study. In order to obtain a betterunderstanding of the disease, human cancer tissue has been studied in aneffort to discover the cause, treatment, prevention and/or diagnosis ofcancer. Early diagnosis of cancer is very important since it increasesthe chances of effecting a complete remission of the disease.

In an effort to utilize known diagnostic tools to detect the presence ofcancer tumors, attempts have been made to demonstrate tumor specificantigens to human carcinomas. These attempts have previously beenunsuccessful with many types of carcinomas since it has not beenpossible to segregate normal tissue antigens from abnormal cancerantigens and demonstrate the specificity of the cancer antigens.

In the efforts to isolate abnormal cancer antigens and demonstrate theirspecificity, attempts have been made to cause the formation of tumorspecific antibodies and demonstrate their presence in sera obtained fromanimals immunized with preparations of human cancer. If consistentlyreproducible, the demonstration of the presence of tumor-specificantibodies in animal antisera would lead to the use of a valuablediagnostic tool.

In order to fully utilize the existence of tumorspecific antibodies inanimal antisera as a diagnostic tool, a test must be developed whichwill demonstrate the presence of the tumor antigen in the blood of thepatient. Procedures which adenocarcinoma been devised have not provenefficient or sensitive in the detection of and differentiation betweencarcinomas origi' nating at different locations within the body, ormetastitic conditions.

Among the possible sources of antigens associated with human carcinomswhich have been most extensively studied by investigators areadenocarcinoma of the colon and digestive tract, meconium, carcinoma ofthe liver, ovarian cysts and carcinnoma of the breast. Sinceadenoocarcinoma of the colon is one of the most widespread cancers andusually requires a surgical procedure for definitive diagnosis, aftersome gross symptomatology has developed, it has been among the mostextensively studied.

Efforts to extract a relatively pure antigen associated with carcinomasor adenocarcinomas have met with either no success or are impracticalfrom a commercial point of view since a process has not been found tomake it possible to completely segregate such an antigen from normaltissue antigens and nonantigenic materials.

The presence of antigens which are stated to be specific toadenocarcinomas of the colon and digestive system by means ofimmunological tolerance and absorption techniques have beendemonatrated, Gold et al.,

.l. Expt. Med. 121 439-462 (1965). However, the prac tical andcommercially feasible isolation of the antigen itself as well as itsassociation with carcinomas and adenocarcinomas had, until the presentinvention, not been achieved.

The tumor-specific antigens have been previously shown to be presentonly iin patients who have adenocarcinoma which originate in digestivesystem epithelium derived from embryonic entodermal tissue, i.e.,esophagus, stomach, duodenom, pancreas and rectum.

It has also previously been demonstrated that the tumorspecific antigensare also present in the digestive organs of fetuses between two and sixmonths of gestation, Gold et al., J. Exptl. Med. 122 467-487 (1965).Thus, for convenience, the antigen has been designed as carcinoembryonicantigen (CEA).

Not only has it heretofore not been possible to isolate and characterizethe CEA by practical rapid methods, but it has not been possible todemonstrate its presence in the blood of persons having adenocarcinomawith a diagnostic test suitable for large screening programs.

DETAILED DESCRIPTION OF THE INVENTION I have discovered that thematerial heretofore known as carcinoembryonic antigen (CEA) is a mixtureof several components, at least two of which have antigenic activitywhich is associated with human carcinoma generally. These two activecomponents are called carcinoembryonic antigen component A (the socalledB antigen) and carcinoembryonic antigen component B (the so-called aantigen).

This invention 'in one significant aspect relates to a method offractionating material having carcinoembryonic antigen activity into itscomponent parts, e.g., carcinoembryonic antigen component A andcarcinoembryonic antigen component B.

This invention in further aspects relates to methods of isolating andcharacterizing the carcinoembryonic antigen components associated withcarcinomas, for diagnostic test procedures and for utilizing eithrradioactive tagged carcinoembryonic antigen material, component A orcomponent B to detect circulating carcinoembryonic antigen material,component A and/or component B.

This invention also relates to a diagnostic test method useful in thedetection of carcinoma and suitable for post-operative monitoring ofcarcinoma patients. Car: cinoma, as used herein, includes all carcinomasand adenocarcinomas present in humans. As used herein, carcinoembryonicantigen material, means the material with carcinoembryonic antigenactivity which contains component A and/or component B.

In order to produce radioactive tagged carcinoembryonic antigenmaterial, component A or component B individually and utilize eachseparately in the improved diagnostic tests of this invention, it isfirst necessary to isolate and purify each entity and confirm itsidentity by means of specific antibodies.

According to this invention, practical processes have been discoveredfor:

a. Isolating, purifying, characterizing and confirming the identify andspecificity of carcinoembryonic antigen material, component A andcomponent B;

b. Utilizing radioactive tagged carcinoembryonic antigen material,component A and/or component B to detect the presence of carcinoma bythe detection of circulating antigens; and

c. Differentiating between circulating free and total carcinoembryonicantigen material, component A and/or component B.

While this invention is concerned with antigens associated withcarcinomas, generally the isolation and purification proceduresdescribed herein will refer to colon carcinoma tissue, metastatic livercarcinoma tissue and meconium all of which are representative of thematerials containiing CEA material, component A and/or component B.

Material having carcinoembryonic antigen activity is isolated andpurified according to the process of this invention by homogenizingadenocarcinoma tissue from primary or metastatic tumors, preferablythose originating within the digestive system, with tumors from thecolon, for example, being suitable, or by homogenizing meconium.

In order to isolate the carcinoembryonic antigen material, component Aand/or component B associated with the homogenized material, it isnecessary to separate all other material from the homogenate, isolatethe carcinoembryonic antigen material and isolate the individualcomponents of the carcinoembryonic antigen material thereof. This isaccomplished by chemical and physical extraction and purificationprocedures. If, for example, only component A or component B are presentin the homogenate, then the isolation and purifica tion procedures willproduce the component without fractionation.

Once the extraction and purification procedures are completed, theidentity of the finally isolated fractions as carcinoembryonic antigenmaterial or a component must be confirmed. This can be accomplished byvarious known techniques, e.g., double diffussion in agar gel,immuno-electrophoresis, hemagglutination, passive cutaneous anaphylaxis,precipitin inhibition and the like.

In order to utilize these techniques, the antibodies used must beconfirmed to be specific for the CEA material, component A and/orcomponent B. Antibodies which meet this criteria can be produced byimmunological tolerance or absorption techniques.

In the absorption technique, tumor antiserum is absorbed with normaltissue and normal fluids (saliva, serum, plasma) in order to removeantibodies produced to normal tissue components. Any residual antibodyactivity in the absorbed antiserum which is directed against tumormaterial is then considered to be tumor specific. This method is notwithout its faults since there is the possibility that tumor specificantibodies may have been removed or inactivated by normal tissuecomponents similar to, but not identical with, the tumor antigens whichinitially stimulated the antibody production.

In the immunological tolerance technique, animals are renderedimmunologically tolerant to normal tissue during neonatal life. Thetolerant animals are then immunized with tumor preparations of the samedonor species. Where adequate suppression of the immune response tonormal tissue components has been achieved, the development ofantibodies apparently specific for the carcinoembryonic antigen activityhas been achieved.

Colon adenocarcinoma tumor tissue and normal colon tissue from the sameindividual can be utilized to illustrate this technique becauseadenocarcinoma of the colon almost never extends submucosally more than6 to 7 cm. on either side of a tumor visible in the gross.

The colon adenocarcinoma tumor tissue and normal colon tissue from thesame individual are treated separately but in parallel fashion. Thetissue is ground up, suspended in a buffer, then homogenized. Thehomogenate is then treated to remove solid particles. Centrifugation orfiltration through successively smaller filter openings are preferred.The purpose is to remove all particles about 0.22 p. or larger, thusremoving all the bacteria present. The supernatant or filtrate isthereby sterilized to insure against bacterial contamination.

Test animals divided into appropriate groups are then immunized with theextracts and, after a suitable time interval, serum is obtained from theanimals. The presence of antibodies in the test sera is demonstrated byeither the Ouchterlony technique of double diffusion in agar gel,immunoelectrophoresis, hemagglutination reactions or passive cutaneousanaphylaxis. The preferred practical method, because of its simplicityand reproducible results is the Ouchterlony technique.

Once the antibodies are demonstrated to be present, it is possible todetermine if a particular extraction technique does, in fact, isolate afraction which contains carcinoembryonic antigen material, component Aor component B.

I have discovered extraction and purification techniques which finallyresult in two separate fractions each of which invariably produces oneprecipitant line in the Ouchterlony technique when tested againstnonabsorbed antisera. The techniques, according to this invention, alsoprovide a means wherein CEA components A and B are separated from eachother and from materials of the same molecular weight and thus areisolated in substantially pure form.

CEA materials as well as components A and B are isolated and purified,according to the preferred process of this invention, from primary ormetastatic carcinoma tissue. Also, CEA material as well as components Aand B can be isolated and purified, according to the process of thisinvention, from embryonic digestive organs of fetuses in the second toseventh month of gestation and from meconiums. The following descriptionwill in most respects be directed to extraction from cancer tissue;however, the process may also apply to embryonic tissue or meconium.

CEA material, CEA component A or CEA component B in either embryonicdigestive organ tissue from the first and second trimester, meconium oradenocarcinoma tissue are extracted with a glycoprotein solvent in whichCEA material, component A and component B are soluble. This is requiredso that precipitable normal proteins and interfering antigenic materialscan be separated from the CEA material or components A and B.Glycoprotein solvents which are suitable are, e.g., perchloric acid,trichloroacetic acid, phosphotungstic acid and the like. However,perchloric acid, because of its availability and ease of use ispreferred.

Prior to the addition of the glycoprotein solvent, the material which isbeing treated is homogenized with water in order to solubilize the CEAmaterial or component A or component B, which ever is present. Theamount of water should be sufficient to solubilize all of thecarcinoembryonic antigen material or component A or component B.Generally, about two liters of water per about every kilogram of treatedmaterial is sufficient. More water can be used, however, it is usuallynot necessary. it is preferred to use distilled water since the chancesof contamination are thereby reduced. The homogenization can be carriedout at from about 4C. to about 60C., however, from about 4C. to aboutroom temperature (about 20C. to about 25C.) is pre' ferred.

The solid particles are then removed from the homogenate. Since the CEAmaterial, component A and component B are water soluble, this can beaccomplished by any convenient method of separation, e.g., filtration orcentrifugation and the like. Centrifugation is preferred because it isfaster and sufficient force can be developed to remove substantially allthe solid particles. Generally, about 3,000 to about 8,000 revolutionsper minute are sufficient to accomplish this. The separation ispreferably carried out at cold temperatures, e.g., about 4C. to aboutC., to prevent loss of activity.

The supernatant from the centrifugation is then treated with aglycoprotein solvent to remove protein materials and interferingantigenic materials.

Any temperature below room temperature is suitable for the addition ofthe glycoprotein solvent to the supernatant of the homogenate.Preferably, however, from about 4C. to about room temperature is used.The temperature of the glycoprotein solvent which is added to thesupernatant can also be variable, preferably, however, the sametemperature as the extracting temperature is utilized. Generally, aconcentrated acid is used as the glycoprotein solvent, e.g., about 0.5Nto about 2N, with 2N being preferred. The solvent is added in aboutequal volume to the supernatant. The time in which the reaction takesplace is usually about 5 to about 30 minutes. Longer times areundesirable since they can result in loss of antigenicity.

A precipitate results. This precipitate is separated from thesupernatant containing the dissolved CEA material, component A orcomponent B. Any convenient method of separation is suitable, e.g.,filtration, centrifugation and the like however, centrifugation underthe same conditions as used with the homogenate is preferred.

Perchloric acid, salts such as sodium chloride and other low molecularweight materials are then removed in order to further purify the system.While it may be possible to accomplish this by precipitating theremaining proteins, I have discovered a fast, efficient methodcomprising dialysis through a semipermeable membrane against apolyethylene glycol with an average molecular weight of about 15,000 to20,000 and a softening point at 60C. A typical suitable commercialproduct useful for this dialysis is M Carbowax marketed by Mann ResearchLaboratories. The dialysis is a critical part of the process since it isfast and efficient and eliminates substantially all diffusible solublematerials except the higher molecular weight materials which include thematerials containing CEA activity. The dialysis is carried out at 4C. to10C., preferably 4C. and is completed in about 18 hours. The process tothis point takes about 24 hours to complete.

The use of the 15,000 to 20,000 molecular weight polyethylene glycol inthe dialysis step is critical to this invention since it aids inspeeding up the isolation of the CEA material, component A or componentB, by the use of only one dialysis step rather than time consumingmultiple dialysis steps against water and eliminates the need for,lyophylizing the retentate.

The resulting retentate is substantially solid in character and containsseveral materials having both higher and lower molecular weights thanthe CEA material, component A or component B.

The separation of the portion of the resulting retentate which containsthe CEA material, or component A or component B to the substantialexclusion of other materials is accomplished according to this inventionby sequential chromatoraphy with two different gel columns followed bychromatography with an appropriate ion exchange column. However,equivalent results are obtained when chromatography on the ion exchangecolumn is used prior to the chromatography on the two different gelcolumns. The eluted fractions from the column chromatography which havea molecular weight of about 200,000-500,000 and a definite peak at thespectrophotmetric absorption wave length of 280 mp. are those containingthe CEA material, component A or component B.

The column chromatography can be accomplished by subjecting theretentate, in solution, to sequential chromatography on two differentgel columns in any order. Practically, however, when using carcinomatissue, a gel column which is used first in accordance with thisinvention is an agarose gel. Agarose is the neutral portion of agar. Thegel material is commercially available from AB Pharmacia, Uppsala,Sweden, under the trade name Sepharose. The gels are available asaqueous suspensions in 0.02% sodium azide as a preservative. The gelstructure is due to hydrogen bonding. The gel is prepared in beaded formhaving a selected particle size and percent agarose. The concentrationof the agarose in the gel determines its fractionation range.

The gels most suitable for use in this invention are those which have aparticle size of from 40 to 210 microns and contain 6 percent by weightagarose. These materials named Sepharose 68" have a fractionation rangewhich separates materials of molecular weight 4 X 10 or less. In theprocess of this invention, Sepharose 6B is used since, when carcinomatissue is used, it permits the separation of the fraction containing theCEA material or its components from extraneous materials ofsubstantially higher or lower molecular weight as well as from colloidalparticles.

The second column contains a gel filter material which is a hydrophilicwater-insoluble cross-linked dextran polymer gel. This matrial and themethod of its manufacture are described in British Pat. No. 854,715. Thegel material, which is commercially avalable from A8 Pharmacia, Uppsala,Sweden, under the name Sephadex, comprises a three dimensionalmacroscopic network of dextran substances bonded or cross-linkedtogether, being capable of absorbing water with swelling. The ability ofthe gel material to take up water is inversely proportional to thedegree of cross-linkage of dextran substances in the gel material. Thegel material is available in a variety of grades differing with respectto degree of porosity. The gel preferred for use in this invention whenchromatography on the ion exchange column follows has an approximatemolecular weight exclusion limit of 100,000, a water regain (g. H O/g.dry gel) of 10 i 1.0, a particle size of 40-120 microns and a bedvolume/mL/g. dry gel of 15-20. The gel is named Sephadex G-100. When thechromatography on the ion exchange column is first then the Seghadex jpreferred for use is Sephadex G-200, having an approximate molecularweight exclusion limit of 200,000, a water regain (g. ihO/g. dry gel) of20: 2.0 a particle size of 40-120 microns and a bed vql l ik it esl 930:50:.

The Sephadexes are employed to further purify the fraction containingthe CEA material or component A or component B. Since the columns havegreater resolving power than the first column (Sepharose) for themolecular weight range of 100,000 to 200,000 for Sephadex 6-100 and150,000 to 250,000 for Sephadex G-200, further separation of the CEAmaterial or component A or component B from lower molecular weightmaterials is achieved. The second column, for all practical purposes,should be used only after the colloidal particles are removed by thefirst column since these particles will clog the column and make itineffective. The problem of colloidal particles is applicable to thetreatment of tumor tissue. However, when, for example, meconium is used,it is preferred to use the Sephadex G-100 or G-200 column first since itremoves bile salts. After the removal of the bile salts, then theSepharose 68 column is advantageously used.

In the preferred process the chromatography is accomplished bydissolving the retentate in aqueous buffer at a pH of from about to 9,preferably pH 7. A typical suitable buffer composition useful in theprocess of this invention is composed of 0.1 M Tris-OH made in 0.135 MNaCl, adjusted to pH 7 with HCl to which 0.02 percent of sodium azide isadded as a preservative. The thus-formed buffer solution is then runthrough the first column, eluted with the same buffer solvent and theeluates collected. The eluates are then dialyzed against thepolyethylene glycol as described above. The collected active fractionsare then redissolved in an aqueous Tris-OH buffer of pH 5 to 9 of thesame composition as described above, the solution is run through thesecond column, eluted with a buffer of pH 5 to 9 of the same compositionas described above and the active fractions are collected and dialyzedas before.

The advantage of utilizing low temperatures, i.e., from about 4C. toabout 10C. is that it maintains stability and can result in increasedresolution. The fractions collected from the second column are thosewhich have a molecular weight of 200,000-500,000 and have a reading witha peak at 280 mu on a UV spectrophotometer. Those fractions collectedfrom the first column are selected based on the same criteria, however,they contain material slightly greater and slightly less (as low as70,000 MW) than 200,000-500,000 MW. The collected fractions contain theCEA material or component A or component B depending on the origin ofthe treated material. This is shown by either the precipitin inhibitionor direct Ouchterlony testing against unabsorbed tumor antiserum. Asingle line precipitate indicates pure CEA activrty.

The active fraction from the second gel column is then subjected tochromatography on an ion exchange column in order to further purify andfractionate the CEA active fraction and separate it from other materialswhich are present. 1 have found that in most cases the fractioncontaining the CEA activity which is derived from colon adenocarcinomatissue from the second gel column contains three different materials(unless, as in some cases of colon adenocarcinoma, only component A orcomponent B are present singly), all

having molecular weights between about 200,000 and 500,000. Of thesematerials, one comprising about 5 percent by weight of the fraction isnon-reactive. A second material, comprising about 10 percent by weightof the fraction has antigenic sites which react with the CEA specificantibody and is identified here as CEA component B. A third material,comprising about percent by weight of the fraction also has antigenicsites which react with the CEA specific antibody and is identified hereas CEA component A.

It has also been found that other materials, e.g., meconium, lungtumors, breast tumors, have different proportions of the components andas a general rule these amounts vary from patient to patient and fromtumor to tumor. Meconium, for example, has only component B.

In order to obtain the pure CEA components it has been found necessaryto utilize an ion exchange column. If only one component is present,then the ion exchange column is used to purify it and confirm itspresence as the sole CEA active material present.

The ion exchange column found suitable for use in accomplishing thedesired separation is a mixed bed column composed of a cation exchanger,carboxymethyl cellulose, and an anion exchanger, diethylaminoethylcellulose. v

The carboxymethyl celluloses most suitable for use in this invention arethose which are microgranular in form, have rod shaped particles with aparticle size distribution expressed as diameter of equivalent sphereswithin a range of about 20 ,u to about 60 [1,, have a capacity of 1.0 i0.1 meq./gm. and a water regain of 2.3-2.7 gm./gm. dry exchanger. Thepreferred ionic form is the Na form. A suitable ion exchanger iscommercially available in a preswollen form from H. Reeve Angel lnc.,Clifton, N.J., under the trade name CM 52.

Another suitable carboxymethylcellulose is CM 32. This does not have thecapacity per volume of CM 52 but is otherwise similar, i.e., it ismicrogranular in form, has rod shaped particles with a particle sizedistribution expressed as diameter of equivalent spheres within a rangeof 20 p. to.60 ,u, has a capacity of 1.0 i 0.1 meq./gm. and a waterregain of 2.3-2.7 gm./gm. dry exchanger. CM 32 is available in dry formfrom H. Reeve Angel Inc., Clifton, NJ.

The diethylaminoethylcelluloses most suitable for use in this inventionare those which are microgranular in form, have rod shaped particleswith a particle size distribution expressed as diameter of equivalentspheres within a range of about 20 [.L to about 60 u, have a capacity of1.0 i 0.1 meq./gm., a water regain of 2.3-2.8 gm./gm. dry exchanger andare in the free base form. A suitable ion exchanger is commerciallyavailable from H. Reeve Angel, lnc., Clifton, NJ. under the trade nameDE 52.

The mixed column is produced by removing the fines from each exchangerby, for example, aspiration of the supernatant resulting from adding a10-fold volume of water, stirring and allowing to settle. Subsequently,a solution made from ammonium acetate in 1.0 M sodium chloride is addedto each column and equal volumes of each of the resulting slurries arethen combined and poured in a 2.5 X 40 cm. column to give a 2.5 X 18 cm.mixed column.

The eluate from the gel columns is dialyzed against the polyethyleneglycol as described above. The resulting material is then dissolved inan aqueous buffered solvent which solubilizes proteins and does not haveaffinity for the column.

A typical suitable solvent is ammonium acetate at pH 4. The bufferedammonium acetate solvent can be formed by adjusting the pH of 0.1 Macetic acid with ammonium hydroxide.

The resulting solution is then clarified. The preferred clarificationmethod is centrifugation which effectively removes all the undissolvedparticles. High speed centrifugation is most effective for thisclarification, preferably at speeds which produce at least 100,000 timesgravity.

The resulting supernatant is then applied to the mixed bed ion exchangecolumn and eluted with an am monium acetate-sodium chloride eluant at pH4. Other allkali metal chloride salts such as potassium chloride arealso suitable. The relative amounts of ingredients in the eluantcompositions are varied. The variations in the composition results in afine separation of the active fraction into its major components andnumerous minor components. This is accomplished by utilizingcompositions containing the ammonium acetate solvent in 0.05, 0.1, 0.25and 1.0 M sodium chloride solutions. The specific relationship of theammonium acetate to the sodium chloride is interrelated to the pH of thesystem, thus, if a different pH is utilized, then the relationship mustbe changed to accomplish the same purpose. The identity and relativeamounts of the major components varies with the identity of theirsource. For example, in a typical case wherein colon carcinoma is thesource of antigen activity, about 85 percent by weight of the materialpresent in the active fraction is eluted when the eluant containsammonium acetate in 0.05 M sodium chloride. This is CEA component A.About 10 percent by weight of the material present in the activefraction is eluted when the eluant contains ammonium acetate in 0.1 Msodium chloride. This is CEA component B. The remaining material iseluted when the eluant contains ammonium acetate in 0.25 M sodiumchloride. ln cases wherein only component A is present or only componentB is present, then the component present will be eluted with the eluantsas described for each component.

An alternative method of producing carcinoembryonic component A and/orcomponent B takes advantage of the fact that it is easier to scale upfor production the mixed bed columns.

The alternative method involves first chromatographing the perchloricacid tumor extract on a mixed bed ion exchange column. The mixed bedcolumn suitable for use is one composed of DE 52 and CM 52 made asdescribed above. In the event both component A and component B arepresent in the tumor extract, they are eluted separately as describedabove, i.e., component A is eluted in solutions having 0.05 M sodiumchloride and component B is eluted in solutions having 0.1 M sodiumchloride. The resulting active components are then chromatographed ontwo different gel columns, the described Sepharose columns preferablySepharose 68 followed by chromatography on a Sephadex column. WhileSephadex G100 is suitable, Sephadex 6-200 provides greaterdifferentiation.

The resulting products are chemically, immunologically andelectrophoretically identical to those resulting from the other processdescribed herein.

In addition to having the same electrophoretic char acteristics as CEAmaterial, i.e., migrating anodally 10-14 cm. in block electrophoresis atthe same time as ferritin marker migrates 18 cm. anodally, using 400volts and about 20mA with a borate buffer of pH 8.6 and ionic strength0.05, CEA component A has a molecular weight of between 120,000 and240,000, is eluted from a mixed bed ion exchange column having thecomposition as described with an ammonium acetate-sodium chloride eluantat pH 4 wherein the eluant contains ammonium acetate in 0.05 M sodiumchloride. Component A also forms a single line precipitate with itsspecific antibody in unabsorbed antiserum in gel diffusion tests, issoluble in perchloric acid and has a spectrophotometer absorption peakwave length of 280 mu.

Also, CEA component B in addition to having the describedelectrophoretic characteristics of CEA material has a molecular weightof between 120,000 and 240,000, is eluted from a mixed bed ion exchangecolumn having the composition as described with an ammoniumacetate-sodium chloride eluant at pH 4 wherein the eluant containsammonium acetate in 0.1 M sodium chloride. Component B also forms asingle line precipitate with its specific antibody in unabsorbedantiserum in gel diffusion tests, is soluble in perchloric acid and hasspectrophotometer absorption peak wave length of 280 mu.

Chemical analysis of component A and component B reveals that theydiffer somewhat in the amounts of amino acids and monosaccharides, forexample, component B has nearly twice as much L-fucose as component Aand about two and one-half times as much sialic acid. Further, there areminor differences in the chemical analysis of the components whenderived from dif fer'ent source s. Typical analyses of the productsproduced by the processes of this invention are as follows:

y. Moles Amino Acid or Monosaccharide Per u Moles Total Amino Acid Thematerial or components containing CEA activity are determined by eitherthe precipitin inhibition or direct Ouchterlony test against unabsorbedtumor antiserum. A single line precipitate indicates pure CEA activity.Thus, any material which forms a single line precipitate with unabsorbedCEA antiserum by either the precipitin inhibition or direct Ouchterlonytechnique of double diffusion in agar gel is included within the scopeof this invention and is suitable for use in the diagnostic testsdescribed herein.

In order to utilize these techniques, the antibodies used must beconfirmed to be specific for CEA material, component A and/or componentB. Antibodies which meet this criteria can be produced by immunologicaltolerance or absorption techniques as mentioned above.

Once the antibodies are demonstrated to be present, it is possible todetermine if a particular extraction technique does, in fact, isolatecarcinoembryonic antigen material, component A or component B. Usingthese techniques, I have found that when the CEA material is present,component A and component B respectively, obtained from the mixed bedion exchanger contains substantially all the CEA activity present in theCEA active fraction. The component which is preferred for use in theradioimmunoassay of CEA is component B. However, either the CEA materialor component A can be satisfactorily utilized.

In another aspect of this invention, l have discovered radioimmunoassaytechniques which are simple to perform and have a high degree ofreproducibility and specificity.

ln radioimmunoassays, it is important that the radio active atom besufficiently reactive with the molecule to be tagged to provide anadequate concentration of radioactivity for determination and theradioactive atom must provide a sufficient number of disintegrations perunit of time to provide sufficient sensitivity for accuratedeterminations. Further, in the case of radioimmunoassay of antigens,the antigenicity must not be delcteriously affected by the conjugationof the radioactive atom to the antigen.

By means of the present invention, it is possible to detect theexistence of human carcinoma growth by assaying a circulating tumorassociated antigen. This invention provides a test sufficientlysensitive to detect at least 1 ng. of CEA material, component A orcomponent B per ml. of serum or plasma. This sensitivity has been foundsufficient to detect abnormal amounts of CEA activity, considered inmost cases to be 2.5 ng. or more. A very minor amount, e.g., less than0.05 ng. of CEA activity may be present in normal situations. Thesensitivity of the assay is limited only by the specific activity of theradioactive atom.

The CEA material, component A or component B can be tagged withradioactive atoms which will react with their chemically reactive groupsand not substantially diminish their antigenicity. I has been found tobe a particularly suitable radioactive atom.

The CEA material, component A or component B can be radioiodinated bymethods known in the art, with minor modifications to concentration andvolumes. The Chloramine T method of Hunter and Greenwood, Biochem. J.91, 46 (1964) using iodine 125 is particularly useful.

A radioiodination efficiency of about to 50 percent can be obtained bythe process described herein. The radioiodination process is equallyapplicable to the CEA material which is purified and isolated prior toits fractionation into components A and B, or each of the components.Preferred for use in this invention, however, is component B.

The reaction is effected, for example, by using a 200 #1. reactionmixture containing 100 pg. of Chloramine T (sodiump-toluenesulfo-chloramine); 0.0250.4 mg.

of CEA material or an individual component thereof and 4 mCi of I in theform of K1 or NaI. The reaction takes place in about 1 minute at roomtemperature and is stopped by the addition of sodium metabisulfite. Thefunction of the Chloramine T is to oxidize the iodide salt to iodine.The function of the sodium metabisulfite is to reduce unreacted I backto its salt. Other reducing agents can also be used, e.g., potassiummetabisulfite. The oxidizing and reducing agents used should not be sostrong that they damage the antigenicity of CEA material or itscomponents. The radioiodinated product can be separated from residual lby chromatography in a cross-linked dextran gel column, e.g., SephadexG-lOO, and removing the tube with the greatest radioactivity in thefirst peak. The resulting product has a specific activity of betweenabout l,0OO-25,000 dpm./ng., preferably between about 10,000 and 20,000dpm./ng., i.e., about 5-10 mp. Ci/ng. of CEA material, component A orcomponent B.

It is necessary, in order to achieve success in the aforesaid diagnostictechnique, to treat the patients blood in a manner which will insurethat all the CEA material, component A or component B, to the exclusionof interfering materials, is in the finally used serum or plasma. Thiscan be accomplished by treating blood serum or plasma from the patientswith a glycoprotein solvent which solubilizes the CEA material,component A or component B, and then clarifying the resulting solution.It has been found that both serum and plasma from the blood of patientsare suitable for use in this process, however, plasma is preferred.

The glycoprotein solvent which has been found suitable for this processis perchloric acid. Perchloric acid of 1.2 M or a sufficient amount toprovide a concentration of about 0.6 M or less of perchloric acid is thepreferred solvent since it removes interfering substances, freesantigenic sites and lowers ionic strength. The resulting solutioncontaining dissolved CEA material,

component A or component B, if any are present, is then clarified. Thepreferred clarification method is to centrifuge, collect the supernatantand dialyze against distilled water, then against buffered water (pH6-6.25, ammonium acetate with 0.01 M acetate). This usually takes about6 to 10 hours. The dialysis residue (retentate) can then be dried bylyophylization, this is not essential however. By using this method apurified extract containing greater than about 95 percent of the CEAmaterial, component A or component B originally pres ent is produced.

It is important to this process that the extract is treated as describedsince the glycoprotein solvent which solubilizes the CEA material,component A or component B in the initial step dissociates anypreexisting CEA-anti-CEA complexes and activates the antigenic sites inthe patients serum or plasma, en-

abling the recovery of substantially all the CEA activity originallypresent. This provides a method for detecting CEA activity in patientswith primary carcinomas and metastatic carcinomas of varying origin.

It is also possible in another aspect of the radioimmunoassay techniquesof this invention to add the antibody directly to the dialyzedsupernatant resulting from the glycoprotein solvent extract of thepatients blood serum or plasma. This eliminates the need for timeconsuming lyophylization procedures and provides a method for detectingCEA materials, component A and/or component B in patients havingcarcinoma.

It is further possible in a preferred aspect of the radioimmunoassaytechniques of this invention to treat the blood serum or plasma bydiluting in such a manner that its ionic strength is reduced, then addthe antibody directly to the dilution.

The dilution can be accomplished by adding at least 100 volumes ofeither water or a salt solution of low ionic strength to each volume ofthe blood serum or plasma. It is preferred to use plasma. Generally anyconvenient salt can be used as long as it does not interfere with thesubsequent treatment with zirconyl phosphate. The salts found suitableare, for example, ammonium acetate, sodium chloride, sodium borate (pH8.4) and the like. Ammonium acetate of 0.01 M or less is preferred.

The dilution of the blood serum or plasma is for the purpose of loweringthe ionic strength of the solution in order to free or activateantigenic sites of any free CEA material or component which is present.This technique does not dissociate any pre-existing CEA- anti-CEAcomplex but makes possible detection of free circulating CEA activity.It is important when using salt solutions as the diluent, that themolarity of the salt be sufficient to lower the ionic strength of theserum or plasma to a level which will activate the antigenic sites.

Since no dialysis procedures are required, this procedure savesconsiderable time and is suitable for initial screening procedures todetect free circulating CEA activity.

Further, in order to effectively conduct the radioimmunoassay, a supplyof antibodies specific to the CEA material, component A and/or componentB must be assured. This is accomplished by immunizing animals with thepurified CEA material or a component in conventional manner as follows.

An emulsifier, e.g., Freunds adjuvant (complete) is added to the CEAmaterial or either component in a saline solution. The emulsion can beinjected in animals intramuscularly, subcutaneously, in the foot pad orany combination of these methods. Animals such as fowl, rabbits, horses,goats, sheep and the like are suitable. The regimen in rabbits, forexample, is injections twice a week until five injections are made.After the last injection, blood is collected from the animal. The serumfrom this blood is unabsorbed CEA antiserum.

In one method, 400 pg. of CEA material or a component in l ml. salinesolution (0.9 percent) is utilized. The injection is madeintramuscularly using a volume about four times that injected in thefood pad.

The antibody present in the antiserum, after absorption with normaltissue components, is specific in its activity against the CEA material,component A and/or component B to the exclusion of other antigens.

In conducting the radioimmunoassay of CEA, procedures based on both thetechniques of isotope dilution and competitive-inhibition can be used.However, the competitive-inhibition method is the preferred method ofthis invention. In these methods, a titration curve, then a standardinhibition curve are obtained.

The standard inhibition curve can be made by the Farr procedure. It is ameasure of the complex formation with specific antibodies. The curvereflects the amount of CEA material, component A and/or component Bpresent per unit of serum. The measurement is in nanograms per ml.,which is plotted against a known percentage of radioactive tagged CEAmaterial, component A or component B. The resulting curve is used toplot the amount of CEA material, component A or component B in apatients serum.

In a preferred method, a standard inhibition curve can also be obtainedby the competitive-inhibition method by adding standard CEA material,component A or component B to a series of tubes containing pow deredperchloric acid extract of normal human serum or plasma. A measuredamount of CEA antiserum which had previously been determined from astandard dilution curve is added to the series of tubes containing adialyzed perchloric acid extract of normal blood serum or plasmadescribed above, or alternatively serum or plasma diluted with 0.01 M(0.0l N) ammonium acetate buffer at pH 6-6.25. In this alternativemethod wherein the test fluid is diluted, a maximum normality of buffershould not be greater than 0.01. Lower normalities are suitable. Whereappropriate, molarity can be used to describe the concentration,equivalent normalities can be calculated by conven' tional means.

The resulting solutions are incubated at about 45C. for a sufficienttime to complete the reaction, usually about 3045 minutes is sufficient.Following the incubation, a measured amount of radioiodinated CEAmaterial, component A or component B is added to each of the tubes. Theincubation is then continued for about an additional 30 minutes at about45C. When the incubation is completed, a precipitant which pre cipitatesthe antibody and antigen-antibody complex but not the antigen, is addedto the solution to coprecipitate the antibody bound CEA material,component A or component B. Preferably, a zirconyl phosphate gel isused.

Under the conditions described above, free CEA material, component A orcomponent B remains in solution, 1 content of the precipitate orsupernatant is then determined from a reading on a suitable instrumentand the amount of CEA material, component A or component B in the serumor plasma is then determined by reference to a standard.

The assay performed on the powdered perchloric acid extracts of serum orplasma processed in the same manner as the standard CEA material,component A or component B, results in a determination of the amount ofCEA material, component A or component B in the patients blood. This inturn is indicative of the presence or absence of carcinoma in thepatient.

According to this invention, the radioimmunoassay can be accomplished byeither a routine isotope dilution procedure or thecompetitive-inhibition assay method described above.

The isotope dilution assay method is carried out by adding a measuredamount of tagged CEA material, component A or component B to aperchloric acid extract of blood serum or plasma which is then dialyzed.The extract is then neutralized with, e.g., NaOH, and a measured amountof antibody is added. The mixture is then dialyzed against thepolyethylene glycol described previously, driving the antibody-antigenreaction to completion.

The resulting precipitate is then dissolved in boric acid buffer at pH6.25. The radioactivity is then determined by adding zirconyl phosphategel to the solution, then centrifuging and assaying the precipitate forradioactivity.

The preferred competitive-inhibition assay method described above iscarried out by dissolving the solid perchloric acid blood serum orplasma extract in a suitable buffered solvent at a pH of 5-8, preferably6.25. While any conventional buffer is suitable, e.g., phosphate buffer,l have found that buffered solvents containing boric acid are preferred.This surprising since heretofore borate buffers have been consideredunsuitable for use in radioimmunoassay or isotope dilution asay at anacid pH. The use of acidic conditions is dietated by the fact that theCEA material, component A or component B are not sufficiently stable atneutral or alkaline pHs to maintain their antigenicity.

A measured amount of antibody is then added to the solution. While anyamount is suitable, 30 units is used for convenience and ease ofmeasurement, however, from about 30 to about 300 units are suitable.

A unit of CEA activity is a nanogram of CEA mate rial, or the equivalentamount of component A or component B. A unit of antibody is the amountof antibody which is bound by a nanogram ofCEA material, or theequivalent amount of component A or component B.

The resulting mixture is then incubated for about 24 hours. 50 Units oftagged CEA material or the equivalent amount of component A or componentB are then added and the mixture is again incubated for about 24 hours.It is possible, however, to use from about to about 500 units, however,20-50 units have been found to be preferred. If there is some CEAmaterial, component A or component B in the serum or plasma, then theamount of unreacted tagged CEA material, component A or component B inthe serum or plasma can be determined either qualitatively orquantitatively. The radioactivity is determined by adding zirconylphosphate gel to the solution, then centrifuging and assaying theprecipitate for radioactivity.

In another preferred aspect of this invention, the assay for determiningfree circulating CEA material, component A or component B is carried outby diluting either blood serum or plasma with at least 100 volumes ofwater or a low ionic salt solution as described previously.

The solution is then transferred into suitable test tubes, 30 units ofCEA antiserum are added and the mixture is incubated at about 45C. for30-45 minutes. 20 to 50 nanograms of radioiodinated CEA material orequivalent amounts of component A or component B, having 10,000 to20,000 dpm./ng. are then added and the mixture is incubated for about 30minutes at about 45C. lf there is some free CEA material, component A orcomponent B, in the blood serum or plasma then the amount of unreactedtagged CEA material, component A or component B in the blood serum orplasma can be determined either qualitatively or quantitatively. Theradioactivity is determined by adding zirconyl phosphate gel to thesolution, then centrifuging and assaying the precipitate forradioactivity.

The method is advantageous because it takes about two hours to complete.It is suitable for determining only the free CEA activity. When used inconjunction with the competitive-inhibition assay methods, it ispossible to have large scale screening or carcinomas.

The following examples illustrate the invention.

EXAMPLE 1 I50 Grams of frozen primary colon adenocarcinoma tumor washomogenized in 5 volumes of distilled water at 5C. for 2 minutes in ahomogenizer. The homogenate was then blended for about 5 minutes in ablender.

The resulting material was then centrifuged for 30 minutes at 5,000 rpm.the supernatent was decanted and a stick was used to prevent the top fatpad which forms from breaking and contaminating the solution. One volumeof 10 percent perchloric acid was added to the supernatant and stirredat 5C. for l0 minutes. The resulting mixture was centifuged for 30minutes at 5,000 rpm. The supernatant was decanted and filtered throughglass wool. The resulting filtrate was then dried by dialysis against a20 M Carbowax solution which was prepared by filling a 10 liter beakerwith 20 M Carbowax Flakes and filling it to the 7 liter mark with aborate buffer at pH 8.4, then stirring until the flakes dissolved. Theresulting solid dissolved in 8 ml. ofTris(hydroxymethyl)-aminomethaneNaCl (Tris-NaCl) solution. The resultingsolution was centrifuged for 30 minutes at 105,000 gravity and 5 ml. ofthe resulting supernatant was applied to a Sepharose 68 column andeluted with the Tris-NaCl solution using drops per tube collected in 5ml. fractions at the rate of 0.5 ml./minute. Tubes 45-57 were pooled andconcentrated by dialysis against 20 M Carbowax. The resultingconcentrate was then applied to a Sephadex G- l 00 column. This waseluted with the Tris-NaCl solution and 4 tubes containing 5 ml. each ofthe first peak were pooled and dried by dialysis against 20 M Carbowax.The resulting solid material was dissolved in 2 ml. of the Tris-NaClsolution and 1 ml. was labelled with I by conventional means. The Ilabelled material was applied to a Sepharose 68 column and eluted withthe Tris-NaCl solution. The pooled fractions 4557 were frozen in 5 ml.tubes and stored at 20C. This is called Tumor Extract No. l (TE-l Whenstudied by gel diffusion versus goat antiserum, a single strong bandappeared. In certain tumor extracts a second minor band appeared.

2 M1. of labelled TE-l were applied to a CM-52zDE- 52 column in 50 ml.of ammonium acetate (pH 4) solution and the column was washed with ml.of ammonium acetate. Almost all the I was retained by the mixed celluoseion exchange column. The column was then eluted with 500 ml. each ofammonium acetate- NaCl solutions containing 0.05 M NaCl, 0.1 M NaCl,0.25 M NaCl, 1.0 M NaCl. Two peaks were eluted with the 0.05 MNaCl-buffer. The first peak is that of CEA component A. The second peakappeared to be degraded l20,000 molecular weight material which iscalled M- 120. A second major peak was eluted with the 0.1 M NaCl-bufferand is pure material having 240,000 molecular weight, it isCEA-component B. A third major peak was eluted with the 0.1 MNaCl-buffer and was not reactive with the antisera. This indicates it isprobably a normal component. Thus, the first peak which was eluted withthe 0.05 M NaCl is CEA component A which contains CEA activity. Thesecond major peak, CEA component B, also contains the CEA activity. Theidentity of the CEA material, component A and component B is confirmedby its forming a single line in the Ouchterlony gel diffusion test withunabsorbed antiserum. When subjected to block electrophoresis usingSephadex G-25 Fine [a cross-linked dextran gel having an approximatemolecular weight exclusion limit of 5,000, a water regain (gH Olg. drygel) of 2.5 1*: 0.2, particle size of 20-80 microns and a bedvolume/ml./g. dry gel of 5] on a non-conductive block, e.g., Lucite, theCEA material, component A and component B behave identically as follows:

The block electrophoresis medium, Sephadex G-25 Fine is swollen withwater for 2 hours at 80C. and washed by decantation with borate of pH8.6 and ionic strength 0.05, then suction filtered through a sinteredglass disk.

A thick slurry of the gel is poured onto a Lucite block suport of 61 cm.X 7.5 cm. X 1 cm. in dimensions and allowed to distribute itself evenlyalong the plate to a depth of 1 cm. The surface is then blotted withcotton gauze sponges until firm but not completely dry.

The block is then fitted with 3 mm. chromatography paper contacts(Whatman) all aligned in the same direction of flow of the paper. Theblock is then placed in the electrophoresis apparatus and allowed toequilibrate for 1 hour under the operating conditions of 400 volts, witha constant current of approximately 20 mA at 4C. A 1 cm. strip is thenremoved from the center of the block and mixed well with a solution of60 mg. of CEA material produced as above, in 0.5 ml. of 0.05 M borate.The resulting slurry is then poured back in the central strip. One totwo drops of ferritin (6 X recrystallized) at a concentration of 100mg./ml. is then spotted at the cathodal extremity of the block. 24 Hoursafter the start of the run, the ferritin marker moves 18 cm. anodally.At that time the block is removed from the electrophoresis apparatus and2 centimeter strips between the zone of application and the anodalextremity are eluted with 2 M NaCl passed through 0.20 a disposable gridmembrane (Nalgene). The activity is localized -14 cm. anodal to theappli cation zone with weaker activity being found 8-10 cm. anodal tothe application zone.

When components A and B are treated separately in the same manner,identical results are obtained.

EXAMPLE 2 500 G. of metastatic liver adenocarcinoma tumor washomogenized in 2 volumes of deionized water at 4C. for 3 minutes in ahomogenizer. The resulting homogenate was centifuged at 7,100 timesgravity for 20 minutes. The supernatant was removed by decantation. Thesupernatant was made 0.6 M with respect to perchloric acid by adding anequal volume of 1.2 M perchloric acid and mixing for 20 minutes at 4C.The resulting mixture was centrifuged for 30 minutes at 5,000 rpm. Theresulting supernatant was collected and neutralized to pH 7.0 by theslow addition of concentrated ammonium hydroxide, with mixing. Theresulting mixture was exhaustively dialyzed against deionized water. Thedialysis extract was concentrated by ultrafiltration with an XM-300membrane to a volume of less than 50 m1. Any particulate matterremaining was removed by centrifuging the concentrated extract at 91,000times gravity for 60 minutes.

The resulting supernatant was chromatographed at pH 4 on a mixed bed ionexchange resin and eluted with a discontinuous sodium chloride gradient.The mixed bed ion exchange resin used was the same one utilized inExample 1, i.e., CM-52zDE-52. The process for the mixed bedchromatography was the same as utilized with labelled TE-l as set forthin Example 1. The active fractions in the 0.05M and 0.1 M sodiumchloride eluents were each separately concentrated and dialyzed to avolume of 4-6 ml. by ultrafiltration using a UM-l O membrane. Each ofthefractions were then further fractionated by gel filtration bychromatographing on a Sepharose 68 column in the same manner as setforth in Example 1. The resulting concentrates of each fraction from the21 1295 ml. fraction were concentrated and dialyzed by ultrafiltrationto a volume of 15 m1. and then applied to a Sephadex G-200 column usingthe same process as set forth for the Sephadex G- column in Example 1.The resulting products were CEA component A which was eluted with 0.05 MNaCl and CEA component B which was eluted with 0.1 M NaCl. Thesematerials were each subjected to electrophoresis and further analyzedimmunologically and chemically and found to be identical to thematerials prepared in Example 1 and identified respectively as componentA and component B.

EXAMPLE 3 Six 5 ml. tubes containing normal serum and six 5 ml. tubescontaining serum from suspected colon cancer patients each wereextracted with an equal volume of 1.2 to 2 molar perchloric acid byshaking for 20 minutes and then centrifuging at 8,000 gravity for 5minutes at 5C. The supernatants were collected and transferred to adialysis tubing and placed in a 250 ml. beaker containing a Carbowaxsolution formed by filling at 10 1 beaker with 20 M Carbowax Flakes andthen filling the beaker to the 7 liter mark with borate buffer of pH 8.4and stirring until the flakes dissolve. After 5 hours of dialysis, theresulting precipitate in the tubes was dissolved in 1 m1. of boratebuffer at pH 6.25 and transferred to 15 X mm. test tubes. 0.1 Ml. ofnormal human serum was added to each tube and mixted. To each of the sixtubes containing normal serum and the perchloric acid extract, 0, 10,50, 100, 250 and 500 nanograms of CEA was added. Then 300 to 500 unitsof CEA antisera was added to each of the 12 tubes and mixed. The tubeswere then stored in an icebox at 5C. for 12 hours. Subsequently, 500units of CEA-l was added to each tube and incubation was continued for18 hours at 5C. Five ml. of zirconyl phosphate gel was added to thetubes and the tubes were then filled with ammonium acetate buffer at pH6.25. The tubes were stopped with rubber stoppers, inverted five timesand centrifuged at 1,500 gravity for 5 minutes. The resultingsupernatant was then discarded. The solid gel which remained was washedwith an ammonium acetate buffer by filling the tubes with the buffer anddispersing the gel with a mixer, then centrifuging at 1,500 timesgravity for 5 minutes. The gel was assayed for bound 1 with a Packard3003 Tri-carb Scintillation spectrometer. Other similar equipment canalso assay for the bound F The results of the serum being tested for CEAactivity were compared to the standard and the amount of CEA material,component A or component B in the unknown sera was determined.

EXAMPLE 4 0, 10, 50, 100 and 500 Nanograms of CEA material standard wereadded to separate tubes each of which contained 5 ml. of normal sera andthen mixed. The standards and serum from suspected cancer patients wereextracted with perchloric acid, centrifuged and dialyzed against 20 MCarbowax in the same manner as in Example 3. The resulting precipitatewas dissolved in 1.0 ml. of borate buffer of pH 6.25, then 500 units ofradioactive tagged CEA material was added, the mixture was mixedthoroughly and then 300 units of CEA antisera were added. The mixturewas dialyzed against a fresh 20 M Carbowax solution and brought todryness in about 2 to 3 hours. The resulting precipitate was dissolvedin 1 ml. of borate buffer of pH 6.25 and then 5 ml. of zirconylphosphate gel were added. The assay for the l was made according to theprocess set forth in Example 3.

EXAMPLE 5 Human meconium is homogenized in 3 volumes of 10 percentperchloric acid at 5C. and centrifuged at 4,000 rpm for 30 minutes. Thesupernatant is then dialyzed against 20 M Carbowax.

The precipitate is taken up in a minimum volume of Tris-NaCl solution ofpH 7 and centrifuged at 105,000 g. for 30 minutes.

5 Ml. of the supernatant is then applied to a Sephadex G-l column andeluted with Tris-NaCl solution. Four tubes of ml. each from the firstfraction were pooled and brought to dryness by dialysis against 20 MCarbowax. The residue is taken up in 8 ml. of NaCl- Tris solution andcentrifuged at 105,000 g. for 30 minutes. 5 M1. of the resultingsupernatant is then applied to a Sepharose 68 column and eluted with theTris- NaCl solution using 80 drops per tube collected in 5 ml. fractionsat the rate of 0.5 ml./minute. Tubes 4557 were pooled and brought to 1ml. by dialysis against 20 M Carbowax. When studied by gel diffusionversus goat antiserum, one strong band developed. It was identical toCEA component B.

EXAMPLE 6 Carcinoembryonic antigen (CEA) material was isolated andradiolabelled with I as described in Example 1.

A goat antiserum mono-specific for CEA material was reacted withradiolabelled CEA material to form an antibody-antigen complex. Theexcess radiolabelled CEA material was separated from the complex byadsorbing the complex with zirconyl phosphate gel (pH 6.25) as describedin Examples 3 and 4.

The radiolabelled CEA was then incubated with the antiserum toillustrate the ion sensitivity of the antigenantibody reaction asfollows:

100 Ng. of radioiodinated CEA material was incubated with antiserumdiluted with water (1-l0,000) at 45C. for 30 minutes in 1 ml. each ofnormal serum (goat, human, rat, rabbit), 0.15 M NaCl, 0.075 M Na- HPO0.15 M Tris-HCl (pH 7.5) and 0.1 M ammonium acetate. This resulted inminimal complex formation.

When the radioiodinated CEA material and the antiserum were incubated in1 ml. each of H 0, 0.01 M NaCl, 0.01 M ammonium acetate 1 percent normalserum diluted in H O, or 0.05 M sodium borate (pH 8.4) antigen-antibodycomplex formation took place.

The antiserum also formed a complex with the radioiodinated CEA materialwhen incubated in 10 ml. of 0.01 M ammonium acetate, 0.1 ml. of normalserum diluted to 10 ml. with water, or 0.005 M sodium borate (pH 8.4).

10 Ng. of CEA material added to dialyzed supernatant from 5 ml. ofnormal serum and 5 m1. of 1 M perchloric acid, neutralized 10 percent ofthe antiserum when incubated at 45C. for 30 minutes prior to theaddition of measured amounts of radioiodinated CEA material.

CEA material was detected in 28 of 30 perehloric acid extracts of serumobtained from patients with colon adenocarcinoma and directly in sera ofmetastatic patients after dilution of 0.1 ml. serum in 10 ml. of water.This indicates that dilution which weakens the ionic strength of theserum provides access to an antigenic site on the CEA material.

EXAMPLE 7 A 3 ml. aliquot of 2 M perchloric acid was added to 5 m1.aliquots of serum of plasma while agitating in a mixer. The mixtureswere allowed to stand at'room temperature for 15 minutes then mixedagain and allowed to settle. The mixtures were centrifuged at 1,000times g. for 5 minutes at room temperature and the supernatants weredialyzed for 36 hours against 25 liters of distilled water at roomtemperature. The dialysis bath was changed five times during a 24 hourperiod. This retentate was then used for testing. All specimens were runin duplicate.

Coat antisera monospecific for CEA material was diluted 1:2000 in 10percent normal human serum and 0.05 M borate buffer pH 8.4. CEA materialwas prepared and labelled with I as described in Example 1.

A dilution curve of the antisera against a constant amount ofradioiodinated CEA material was carried out in the dialysates ofperchloric acid serum extracts to which 1 m1. of borate buffer (0.05 sM,pH 8.4) was added.

Six tubes of the serum were placed in a water bath at 45C. for one-halfhour. After incubation of the mixture, 5 ml. of ammonium acetatesolution (0.1 M, pH 6.25) prepared by adjusting the pH of 0.1 M aceticacid to 6.25 with cone. NH OH, and 4 ml. of zirconyl phosphate gel (pH6.25) were added to each tube and the tubes capped and inverted severaltimes. The tubes were then centrifuged at 3,000 rpm for 15 minutes andthe supernatants were discarded. The residue from each tube wasresuspended in 10 ml. of ammonium aeetate solution, recovered bycentrifugation and assayed for bound 1 in a gamma scintillation counter.

A titration curve was carried out by adding known amounts of unlabelledCEA material to the retentates of serum perchloric acid extracts. 1 M1.borate buffer (pH 8.4, 0.5 M) and 0.5 m1. ofa 1:1000 dilution ofantisera was added to each of 6 tubes and incubated in a water bath at45C. for /2 hour. Then 0.1 ml. of radioiodinated CEA material containing24,000 DPM was added to each specimen. The tubes were mixed well andreincubated for one-half hour. 5 M1. of ammonium acetate solution (0.1 MpH 6.25) and zirconyl phosphate gel (pH 6.25) were then added to eachtube. The tubes were capped, inverted several times and centrifuged at1200 times g. for 15 minutes at room temperature. The supernatant wasdiscarded and the gel precipitate was resuspended in 10 ml. ammoniumacetate solution (0.1 M, pH 6.25). The gel was separated bycentrifugation and assayed for bound P Specimens from patients were runin exactly the same manner as above except that unlabelled antigen wasnot added.

About of the I labelled material reacted with the antisera. A finaldilution of 1110,000 of the CEA- antisera in the perchloric acidextracts from serum reacts with 70 percent of the maximum amount of1abelled antibody reactive material. The 1110,000 dilution was thereforeutilized.

Incubation of the antisera diluted with water (1:l(),000) withunlabelled CEA material prior to addition of labelled antigen shows thatthe reaction of antibody with antigen is linear at antigenconcentrations of from 1.5 to 10 ng./ml. but that the reaction is lesssensitive at concentrations above 20 ng./ml.

Of 487 patients tested, those with carcinomas of the breast, lung colonhad detectable concentrations of CEA material in their serum.

Of 229 patients without malignant disease, 11 had detectable antigen intheir serum. Two of these later developed cancer, one had adenomatiouspolyp of the colon and had severe emphysema.

EXAMPLE 8 1 Ml. of plasma from suspected cancer patients was dilutedwith 4 ml. of physiological saline solution. An equal volume of 1.2molar perchloric acid was added and the mixture was agitated for 20minutes then centrifuged at 8,000 times gravity for 5 minutes at roomtemperature. The supernatant was collected and transferred to a dialysistubing and dialyzed overnight against distilled water. The resultingretentate was dialyzed against an ammonium acetate solution of pH 6 to6.25 containing 0.01 molar acetate, for 3 hours at room temperature. Theretentate was transferred into 20 ml. test tubes. 30 Units of CEAantiserum was then added and the mixture incubated for 30 to 45 minutesat 45C. 50 Ng. of CEA-l containing 10,000 to 20,000 dpm./ng. was thenadded and the mixture incubated for 30 minutes at 45C. 5 MI. of pH 6.25zirconyl phosphate gel was added to each test tube and 5 ml. of ammoniumacetate solution (pH 6.25, 0.1 M) were then added. After mixing thetubes were centrifuged at 1,500 times gravity for 5 minutes at roomtemperature and the resulting supernatant was discarded. The solid gelwhich remained was washed with ammonium acetate buffer by filling thetubes with the buffer and dispersing the gel with a mixer, thencentrifuging at 1,500 times gravity for 5 minutes. The gel was assayedfor bound 1 with a Packard 3003 Tri-carb Scintillation Spectrometer. 1fCEA is present in the plasma, the amount of bound CEA-l will be reducedproportionately.

EXAMPLE 9 10 Ml. of water were added to 0.1 ml. of plasma in a ml. testtube. 30 Units of CEA antiserum were then added and the mixtureincubated for 30-45 minutes at 45C. 50 Ng. of CEA-l containing 10,000 to20,000 dpm./ng. were then added and the mixture incubated for 30 minutesat 45C. 5 M1. of pH 6.25 zirconyl phosphate gel and 5 ml. of ammoniumacetate solution (pH 6.25, 0.01 M) were then added to each test tube.After mixing, the tubes were centrifuged at 1,500 times gravity for 5minutes at room temperature and the resulting supernatant was discarded.The solid gel which remained was washed with the ammonium acetate bufferby filling the tubes with the buffer and dis persing the gel with amixer, then centrifuging at 1,500 times gravity for 5 minutes. The gelwas assayed for bound 1 with a Packard 3003 Tri-carb ScintillationSpectrometer. 1f CEA is present in the plasma, the amount of bound CEA-lwill be reduced accordingly.

I claim:

1. A process for producing human carcinoembryonic antigen component Awhich comprises a. homogenizing adenocarcinoma tissue;

b. separating the solid particles from the homogenate;

c. treating the supernatant with a glycoprotein solvent in which thecarcinoembryonic antigen component A is soluble;

d. separating the precipitate and clarifying the resulting solution;

e. dialyzing the clarified solution against a polyethylene glycol withan average molecular weight of about 15,000 to 20,000 and a softeningpoint at 60C.;

f. dissolving the retentate in an aqueous buffer of pH from about 5 to9;

g. subjecting the resulting material to sequential chromatography on twodifferent gel columns, the first of which is an agarose gel having about6 percent by weight agarose and a particle size of from 40 to 210microns and the second of which is a hydrophilic water-insolublecross-linked dextran polymer gel;

h. collecting and concentrating the eluates which have aspectrophotometric absorption peak wave length of 280 mp. and amolecular weight of about 200,000 to 500,000;

i. subjecting the resulting eluates to chromatography on a mixed bedcellulose ion exchange column comprising microgranular diethylaminoethylcellu lose in the free base form having rod shaped particles, a capacityof 1.0 1*: 0.1 meq./gm., a particle size distribution of about 20 ,u. toabout 60 p. and a water regain of 2.3 to 2.8 gm/gm. dry exchanger and amicrogranular carboxymethylcellulose with a capacity of 1.0 0.1 meq/gm.having rod shaped particles, aparticle size distribution of about 20 ,u.to about 60 ,u. and a water regain of 2.3 to 2.7 gm./gm. exchanger;

j. collecting the eluates resulting from eluting with ammonium acetateat pH 4 in 0.05 M sodium chloride.

2. The process of claim 1 wherein prior to step (e) and subsequent tostep (d) the clarified solution is dialyzed against distilled water.

3. The process of claim 1 wherein in step (g) the material is firstdissolved in an aqueous buffer of about pH 7 then run through theagarose gel column which separates the CEA material, component A orcomponent B containing fraction from higher and lower molecular weightmaterial and colloidal particles, the eluates having a molecular weightranging from about 200,000 500,000 and a reading with a peak at 280 my.on a UV spectrophotometer are collected, dialyzed against polyethyleneglycol of molecular weight about 20,000, the retentate dissolved in anaqueous buffer of pH about 7 and run through a cross-linked dextran gelcolumn which has a higher resolving power than the first column andseparates CEA material, component A or component B containing fractionfrom higher and lower molecular weight material.

4. The process of claim 1 wherein in step (i) the eluate is dialyzedagainst polyethylene glycol of molecular weight of about 20,000, theretentate dissolved in an aqueous low ionic strength buffer of pH about4, then run through said mixed bed ion exchange column.

5. The process of claim 1 wherein in step (i) the eluate is dialyzedagainst polyethylene glycol of molecular weight of about 20,000, theretentate is dissolved in an aqueous ammonium acetate buffer with 0.1 Macetate of pH about 4, then run through said mixed bed ion exchangecolumn.

6. A process for producing human carcinoembryonic antigen component Bwhich comprises a. homogenizing adenocarcinoma tissue;

b. separating the solid particles from the homogenate;

c. treating the supernatant with a glycoprotein solvent in which thecarcinoembryonic antigen component B is soluble;

d. separating the precipitate and clarifying the resulting solution;

e. dialyzing the clarified solution against a polyethyl ene glycol withan average molecular weight of about 15,000 to 20,000 and a softeningpoint at 60C.;

f. dissolving the retentate in an aqueous buffer of pH from about to 9;

g. subjecting the resulting material to sequential chromatography on twodifferent gel columns, the first of which is an agarose gel having about6 percent by weight agarose and a particle size of from 40 to 210microns and the second of which is a hydrophilic water-insolublecross-linked dextran polymer gel;

h. collecting and concentrating the eluates which have aspectrophotometric absorption peak wave length of 280 mp. and amolecular weight of about 200,000 to 500,000;

i. subjecting the resulting eluates to chromatography on a mixed bedcellulose ion exchange column comprising microgranular diethylaminoethylcellulose in the free base form having rod shaped particlcs, a capacityof 1.0 i 0.1 meq./gm., a particle size distribution of about t to about60 u and a water regain of 2.3 to 2.8 gmjgm. dry exchanger and amicrogranular carboxymethycellulose with a capacity of 1.0 i 0.1 meqjgmhaving rod shaped particles, a particle size distribution of about 20 p.to about 60 ,u. and a water regain of 2.3 to 2.7 gm./gm. exchanger;

j. collecting the eluates resulting from eluting with ammonium acetateat pH 4 to 0.1 M sodium chloride.

7. The process of claim 6 wherein in step (g) the material is firstdissolved in an aqueous buffer of about pH 7 then run through theagarose gel column which separates the CEA material, component A orcomponent B containing fraction from higher and lower molecular weightmaterial and colloidal particles, the eluates having a molecular weightranging from about 200,000 500,000 and a reading with a peak at 280 my.on a UV spectrophotometer are collected, dialyzed against polyethyleneglycol of molecular weight about 20,000, the retentatee dissolved in anaqueous buffer of pH about 7 and run through a cross-linked dextran gelcolumn which has a higher resolving power than the first column andseparates CEA material, component A or component B containing fractionfrom higher and lower molecular weight material.

8. The process of claim 6 wherein in step (i) the eluate is dialyzedagainst polyethylene glycol of molecular weight of about 20,000, theretentate dissolved in an aqueous buffer of pH about 4, then run throughsaid mixed bed ion exchange column.

9. The process of claim 6 wherein in step (i) the eluate is dialyzedagainst polyethylene glycol of molecular weight of about 20,000, theretentate is dissolved in an aqueous ammonium acetate buffer with 0.1 Macetate of pH about 4, then run through said mixed bed ion ex- UNITEDSTATES PATENT AND TRADEMARK OFFICE CETIFICATE OF CORRECTION PATENT NO.3,867,363

DATED I February 18, 1975 lNV'ENTORQ) HANS JOHN HANSEN It is certifiedthat error appears in the above-identified patent and that said LettersPatent are hereby corrected as shown below:

Title page after No. [76] insert [73] Assignee: Hoffmann-La Roche .Inc.Nutley, New Jersey Column 24, line 7 "pH 4 to 0. l M" should be pH 4 in0.1 M

Signed and Scaled this Thirty-first Day of August 1976 [SEAL] Arrest:

RUTH C. MASON C. MARSHALL DANN Arresting Officer Commissioner nj'Parenrsand Trademarks UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OFCORRECTION PATENT NO. I 3,867,363

DATED 1 February 18, 1975 |NVENTOR(S) HANS JOHN HANSEN it is certifiedthat error appears in the above-identified patent and that said LettersPatent are hereby corrected as shown below:

Title page after N0. [76] insert [73] Assignee: Hoffmann-La Roche Inc.Nutley, New Jersey Column 24, line 7, "pH 4 to 0.1 M" should be pH4in0.1M

Signed and Scaled this RUTH C. MASON Arresting Officer C. MARSHALL DANNCommissioner nj'larenrs and Trademarks

1. A PROCESS FOR PRODUCING HUMAN CARCINOEMBRYONIC ANTIGEN COMPONENT AWHICH COMPRISES A. HOMOGENIZING ADENOCARCINOMA TISSUE, B. SEPARATING THESOLID PARTICLES FROM THE HOMOGENATE, E. TREATING THE SUPERNATANT WITH AGLYCOPROTEIN SOLVENT IN WHICH THE CARCINOEMBRYONIC ANTIGEN COMPONENT AIS SOLUBLE, D. SEPARATING THE PRECIPITATE AND CLARIFYING THE RESULTINGSOLUTION, E. DIALYZING THE CLARIFIED SOLUTION AGAINST A POLYETHYLENEGLYCOL WITH AN AVERAGE MOLECULAR WEIGHT OF ABOUT 15,000 TO 20,000 AND ASOFTEINING POINT AT 60*C, F. DISSOLVING THE RETENTATE IN AN AQUEOUSBUFFER OF PH FROM ABOUT 5 TO 9, G. SUBJECTING THE RESULTING MATERIAL TOSEQUENTIAL CHROMATOGRAPHY ON TWO DIFFERENT GEL COLUMNS, THE FIRST OFWHICH IS AND AGAROSE GEL HAVING ABOUT 6 PERCENT BY WEIGHT AGAROSE AND APARTICLE SIZE OF FROM 40 TO 210 MICRONS AND THE SECOND OF WHICH IS AHYDROPHILIC WATER-INSOLUBLE CROSSLINKED DEXTRAN POLYMER GEL, H.COLLECTING AND CONCENTRATING THE ELUATES WHICH HAVE A SPECTROPHOTOMETRICABSORPTION PEAK WAVE LENGTH OF 280 M$ AND A MOLECULAR WEIGHT OF ABOUT200,000 TO 500,000, I. SUBJECTING THE RESULTING ELUATES TOCHROMATOGRAPHY ON A MIXED BED CELLULOSE ION EXCHANGE COLUMN COMPRISINGMICROGRANULAR DIETHYLAMINOETHYL CELLULOSE IN THE FREE BASE FROM HAVINGROD SHAPED PARTICULES, A CAPACITY OF 1.0 $0.1 MEQ./GM, A PARTICLE SIZEDISTRIBUTION OF ABOUT 20 $ TO ABOUT 60$ AND A WATER REGIN OF 2.3 TO2.8GM/GM. DRY A MIXED BED CELLULOSE ION EXCHANGE COLUMN COMPRISING WITHA CAPACITY OF 1.0$0.1 MEQ/GM. HAVING ROD SHAPED PARTICLES, A PARTICLESIZE DISTRIBUTION OF ABOUT 20 $ TO ABOUT 60$ AND A WATER REGIAN OF 2.3TO 2.7 GM./GM. EXCHANGER, J. COLLECTING THE ELUATES RESULTING FROMELUTING WITH AMMONIUM ACETATE AT PH 4 IN 0.05 M SODIUM CHLORIDE.
 2. Theprocess of claim 1 wherein prior to step (e) and subsequent to step (d)the clarified solution is dialyzed against distilled water.
 3. Theprocess of claim 1 wherein in step (g) the material is first dissolvedin an aqueous buffer of about pH 7 then run through the agarose gelcolumn which separates the CEA material, component A or component Bcontaining fraction from higher and lower molecular weight material andcolloidal particles, the eluates having a molecular weight ranging fromabout 200,000 -500,000 and a reading with a peak at 280 m Mu on a UVspectrophotometer are collected, dialyzed against polyethylene glycol ofmolecular weight about 20,000, the retentate dissolved in an aqueousbuffer of pH about 7 and run through a cross-linked dextran gel columnwhich has a higher resolving power than the first column and separatesCEA material, component A or component B containing fraction from higherand lower molecular weight material.
 4. The process of claim 1 whereinin step (i) the eluate is dialyzed against polyethylene glycol ofmolecular weight of about 20,000, the retentate dissolved in an aqueouslow ionic strength buffer of pH about 4, then run through said mixed bedion exchange column.
 5. The process of claim 1 wherein in step (i) theeluate is dialyzed against polyethylene glycol of molecular weight ofabout 20,000, the retentate is dissolved in an aqueous ammonium acetatebuffer with 0.1 M acetate of pH about 4, then run through said mixed bedion exchange coLumn.
 6. A process for producing human carcinoembryonicantigen component B which comprises a. homogenizing adenocarcinomatissue; b. separating the solid particles from the homogenate; c.treating the supernatant with a glycoprotein solvent in which thecarcinoembryonic antigen component B is soluble; d. separating theprecipitate and clarifying the resulting solution; e. dialyzing theclarified solution against a polyethylene glycol with an averagemolecular weight of about 15,000 to 20, 000 and a softening point at60*C.; f. dissolving the retentate in an aqueous buffer of pH from about5 to 9; g. subjecting the resulting material to sequentialchromatography on two different gel columns, the first of which is anagarose gel having about 6 percent by weight agarose and a particle sizeof from 40 to 210 microns and the second of which is a hydrophilicwater-insoluble cross-linked dextran polymer gel; h. collecting andconcentrating the eluates which have a spectrophotometric absorptionpeak wave length of 280 m Mu and a molecular weight of about 200,000 to500,000; i. subjecting the resulting eluates to chromatography on amixed bed cellulose ion exchange column comprising microgranulardiethylaminoethyl cellulose in the free base form having rod shapedparticles, a capacity of 1.0 + or - 0.1 meq./gm., a particle sizedistribution of about 20 Mu to about 60 Mu and a water regain of 2.3 to2.8 gm./gm. dry exchanger and a microgranular carboxymethycellulose witha capacity of 1.0 + or - 0.1 meq./gm having rod shaped particles, aparticle size distribution of about 20 Mu to about 60 Mu and a waterregain of 2.3 to 2.7 gm./gm. exchanger; j. collecting the eluatesresulting from eluting with ammonium acetate at pH 4 to 0.1 M sodiumchloride.
 7. The process of claim 6 wherein in step (g) the material isfirst dissolved in an aqueous buffer of about pH 7 then run through theagarose gel column which separates the CEA material, component A orcomponent B containing fraction from higher and lower molecular weightmaterial and colloidal particles, the eluates having a molecular weightranging from about 200,000 - 500,000 and a reading with a peak at 280 mMu on a UV spectrophotometer are collected, dialyzed againstpolyethylene glycol of molecular weight about 20,000, the retentateedissolved in an aqueous buffer of pH about 7 and run through across-linked dextran gel column which has a higher resolving power thanthe first column and separates CEA material, component A or component Bcontaining fraction from higher and lower molecular weight material. 8.The process of claim 6 wherein in step (i) the eluate is dialyzedagainst polyethylene glycol of molecular weight of about 20,000, theretentate dissolved in an aqueous buffer of pH about 4, then run throughsaid mixed bed ion exchange column.
 9. The process of claim 6 wherein instep (i) the eluate is dialyzed against polyethylene glycol of molecularweight of about 20,000, the retentate is dissolved in an aqueousammonium acetate buffer with 0.1 M acetate of pH about 4, then runthrough said mixed bed ion exchange column.